CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority of
U.S. Provisional Patent Application 62/081,825 filed on November 19, 2014 in the name of Kiromic LLC titled. METHODS FOR PRODUCTION AND USE OF A NOVEL NANOPARTICLE-BASED
VACCINE TARGETING CANCER/TESTIS ANTIGENS (CTA) IN SOLID AND HEMATOLOGICAL MALIGNANCIES
The entire disclosure of the provisional application is incorporated by reference
herein for all purposes.
BACKGROUND OF THE DISCLOSURE
Technical Field
[0002] The present disclosure relates to the field of anti-cancer vaccines, More particularly,
the present invention relates to the field of oral anticancer vaccines wherein one
or more of the vaccine ingredients are embedded in a matrix in the form of nanoparticles
which protects proteinaceous ingredients from gastric attack.
Description of the Related Art
[0003] Immunotherapy strategies such as cancer vaccines are considered less toxic and more
specific than current treatments available to cancer patients. Due to their specificity,
as well as to potent and lasting effects and applicability to virtually any tumor
type, anti-cancer vaccines are a focus of interest of clinical oncologists. A critical
step in the development of cancer vaccines is the implementation of vaccination strategies
that allow for consistent induction of immune responses to tumor antigens (
Chiriva-Internati, M et al. PLoS One. 5(5):e10471 (2010);
Stagg et al. Methods Mol Med. 64:9-22 (2001). However, despite promising therapeutic potential, there is still a need to overcome
major challenges in cancer immunotherapy, including poor immunogenicity of cancer
vaccines, off-target side effects of immunotherapeutics, as well as suboptimal outcomes
of adoptive T cell transfer-based therapies.
SUMMARY OF THE DISCLOSURE
[0004] Disclosed is a vaccine composition comprising:
- a. particles comprising nanoparticles, microparticles or a mixture thereof, the particles
comprising a gastric acid resistant, enteric erodable matrix comprising beta cyclodextrin
and hydroxypropylmethyl cellulose acetate succinate (HPMCAS), the particles encapsulating
one or more cancer testis antigens (CTA), and an M-cell targeting lectin such as aleuria
aurantia lectin (AAL), the lectin being present in a microfold cell (M-cell) targeting
effective amount; and
- b. An aqueous vehicle in which said particles are suspended,
wherein the one or more CTA is present in said vaccine composition in an amount effective
to stimulate, upon oral administration of said vaccine composition to a subject having
a malignant tumor expressing the one or more CTA, an immune response against said
tumor.
[0005] In some embodiments, the CTA is provided in an amount within the range from about
1 to about 10% w/w based on the weight of the particles. In some embodiments, the
one or more CTA is selected from the group consisting of SP17, AKAP4, PTTG1, and Ropporin
1 and combinations of two or more of the foregoing. In other embodiments, the one
or more CTA is selected from the group consisting of ASP, MAGE, NY-ESO-1, HER2neu,
and Hm1.24 and combinations of two or more of the foregoing. In some embodiments,
the one or more CTA is selected from the group consisting of SP17, AKAP4, PTTG1, and
Ropporin1, ASP, MAGE, NY-ESO-1, HER2neu, and Hm1.24and combinations of two or more
of the foregoing.
[0006] In further embodiments, the lectin is AAL. In more specific embodiments, the AAL
is provided in an amount within the range from about 0.1 to about 0.5% (w/w) based
on the weight of the particles.
[0007] In some embodiments, the vaccine composition further comprises at least one adjuvant.
[0008] In some embodiments, the at least one adjuvant has the property of stimulating at
least a Th1 cellular immune response; In further embodiments, the at least one adjuvant
has the property of stimulating both a Th1 and a Th2 immune response. In more specific
embodiments, the at least one adjuvant comprises a first adjuvant for stimulating
at least a Th1 immune response and a second adjuvant for stimulating a Th2 immune
response. In further specific embodiments, the at least one adjuvant is selected from
the group consisting of CpG, MF59, alum, flagellin, R848, monophosphoryl lipid A,
ODN 1826, and combinations of at least two of the foregoing.
[0009] In some embodiments, the adjuvant comprises CpG.; In more specific embodiments, the
CpG is present in an amount within the range from about 0.1 to about 0.5 % (w/w) based
on the weight of the particles. In further embodiments, the adjuvant is provided in
an amount within the range from about 0.1 to about 0.5 % (w/w) based on the weight
of the particles.
[0010] In some embodiments of the vaccine composition, the matrix also comprises ethyl cellulose.
[0011] In some embodiments, the vaccine composition further comprises at least one immune
response stimulating cytokine. In more specific embodiments, the at least one cytokine
is selected from the group consisting of interleukin 2 (IL-2), interleukin 12 (IL-12)
or both.
[0012] In some embodiments, the vaccine composition further comprises a lysate from said
tumor. In more specific embodiments, the lysate is present in an amount providing
a lysate protein content (as measured by BCA assay) to the particles within the range
from about 1 to about 10 % (w/w) based on the weight of the particles. In further
embodiments, the lysate has been prepared by a process comprising use of hypotonic
lysis buffer.
[0013] In some embodiments, the matrix comprises 60% beta cyclodextrin, 20% HPMCAS and 15%
ethyl cellulose. In other embodiments, the matrix comprises 60% beta cyclodextrin.
[0014] In some embodiments, the vaccine composition comprises CTA and AAL (or other lectin)
are embedded in the matrix. In some embodiments, the CTA, the AAL (or other lectin)
and the adjuvant are embedded in the matrix. In further embodiments, the CTA, the
AAL (or other lectin), the adjuvant and the cytokine are embedded in the matrix.
[0015] In some embodiments, the particles of the vaccine composition or of a particle assembly
have an average diameter based on particle volume within the range from about 1 to
about 5 micrometers. In some embodiments, the particles have a normal size distribution
with at least 90 percent of the particles having an average diameter between about
0.5 and 5 micrometers. In some embodiments, the the particles have a zeta potential
within the range from about +50 to about -50 mV.
[0016] In some embodiments, the vaccine composition comprises a CTA selected from one or
more of MAGE-A3, MAGE-A9 and MAGE-A12.
[0017] In another aspect, the present disclosure relates to an assembly of particles comprising
nanoparticles, microparticles or mixtures thereof for use in a vaccine composition
comprising:
- a. a polymer matrix comprising beta cyclodextrin and hydroxypropylmethyl cellulose
acetate succinate (HPMCAS),
- b. one or more cancer testis antigens (CTA);
- c. aleuria aurantia lectin (AAL) or another M cell targeting lectin,
and optionally one or more of the following;
d. an immune response stimulatory cytokine;
e. an adjuvant; and,
f. tumor lysate antigens.
[0018] In some embodiments, the matrix comprises 60% beta cyclodextrin. In some embodiments,
the matrix comprises 30% HMPCAS and 15% EC. In further embodiments, the CTA is one
or more antigens selected from the group consisting of SP17, AKAP4, PTTG1, Ropporin,
ASP, MAGE, NY-ESO-1, HER2neu, and Hm1.24 and combinations of two or more of the foregoing.
[0019] In some embodiments, the particles according comprise an adjuvant selected from the
group consisting of CpG, MF59, alum, flagellin, R848, monophosphoryl lipid A, ODN
1826, and combinations of at least two of the foregoing.
[0020] In some embodiments, the cytokine in the particles is at least one cytokine that
has the property of stimulating at least one of Th1 and Th2 immune responses. In further
embodiments, the cytokine in the particles is one or both of IL-2 and IL-12.
[0021] In some embodiments of the particle assembly, the particles further comprising a
lysate from the tumor of a subject suffering from a malignant tumor. In more specific
embodiments, the particles have an average diameter based on particle volume within
the range from about 1 to about 5 microns. In some embodiments the particles in the
assembly have a normal size distribution with at least 90 percent of the particles
having an average diameter between about 0.5 and 5 micrometers. In further embodiments,
the particles have a zeta potential within the range from about -50 to about +50 mV.
[0022] In some embodiments of the particle assembly, the CTA is provided in an amount effective,
upon oral administration of the nanoparticles in a vaccine composition to a subject
having a malignant tumor expressing said at one or more CTA, to stimulate an immune
response against the tumor.
[0023] In some embodiments of the particle assembly, the AAL or other lectin is provided
in an amount effective to recruit microfold cells of said subject.
[0024] In some embodiments of the particle assembly, the at least one cytokine is IL-2 and
IL-12.
[0025] In some embodiments of the particle assembly, the at least one adjuvant is CpG.
[0026] Other embodiments of the particle assembly have one or more of the features recited
for the particles in the vaccine compositions above.
[0027] In some embodiments of the particle assembly, the CTA is one or more of MAGE-A3,
MAGE-A9 and MAGE-A12. In other embodiments, the matrix also comprises crosslinked
ethyl cellulose in addition to beta cyclodextrin. In further embodiments, the matrix
comprises 60% beta cyclodextrin, 20% HPMCAS and 15% ethyl cellulose.
[0028] In yet another aspect, the present disclosure relates to a method for treating cancer
comprising administering to a subject afflicted with a malignant tumor a vaccine composition
comprising particles comprising microparticles, nanoparticles or mixtures thereof,
wherein the particles comprise an enteric erodable matrix comprising beta cyclodextrin,
hydroxypropylmethyl cellulose acetate succinate (HPMCAS), and optionally ethyl celluloe,
the particles carrying one or more cancer testis antigens (CTA) expressed in the tumor,
and an M cell targeting lectin, aleuria aurantia lectin (AAL) or another M cell targeting
lectin, the AAL or other lectin being present in a microfold cell targeting effective
amount; and
- a. An aqueous vehicle in which said particles are suspended,
wherein the CTA is present in said vaccine composition in an amount effective to stimulate,
upon oral administration of said vaccine composition to a subject having a malignant
tumor, an immune response against said tumor, and thereby treating the tumor.
[0029] In some embodiments, the method comprises repeating the administration at least two
and up to 10 times at an interval of 1 to 3 days after the preceding administration.
[0030] In some embodiments of the method, the treatment results in one or more of reducing
tumor burden in said subject and prolonging survival of said subject.
[0031] In embodiments of the method, the subject is afflicted with a malignant tumor selected
from the group of solid tumors and hematologic malignancies. In further embodiments,
the tumor is a solid tumor selected from the group consisting of ovarian cancer, hepatocellular
carcinoma, breast cancer, and prostate cancer or any other solid tumor disclosed in
the present specification.
[0032] In alternative embodiments, the tumor is a hematologic malignancy selected from the
group consisting of multiple myeloma, acute myelogenous leukemia, acute lymphoblastic
leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia
or any other hematologic tumor disclosed in the present specification.
[0033] In further embodiments of the method the administered vaccine has one or more of
the features recited above for the aspect of the present disclosure directed to a
vaccine composition. In further embodiments, the method comprises administering to
the subject a vaccine composition in an amount within the range between about 5mg
to 320 mg said composition containing from about 1 to about 10 % (w/w) of CTA, from
about 0.1 to about 0.5 % (w/w) of AAL based on the weight of the particles.
[0034] In more specific embodiments, the tumor is ovarian cancer.
[0035] In more specific embodiments, CTA is SP17.
[0036] In some embodiments of the method, the composition further comprises an adjuvant.
In some embodiments of the method, the composition further comprises at least one
immunostimulatory cytokine is two cytokines: IL-2 and IL-12.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037]
Figure 1 is a schematic representation of steps in microparticle preparation.
Figure 2 is a bar graph of nitrite (stable physiological reservoir of nitric oxide)
release from the dendritic cells (DC) following incubation with respective formulations
for 24 hours. The star above the bar indicates statistical significance (p<0.05).
All adjuvant treated groups included 7µg of SP17 in microparticles (MP) and 2µg of
antigen in MP.
Figure 3 is a bar graph of mean fluorescence intensity indicating CD80 expression
on DCs following incubation of microparticle formulations for 16 hours in various
adjuvants. Vaccine mp (Vaccine microparticle alone), VM (Vaccine + MF59), VP (Vaccine
+ P4), VR (Vaccine + R848), VMR (Vaccine+ MF59 + R848), VAM (Vaccine + Alum + MF59),
VMP (Vaccine + MF59 + P4), VPR (Vaccine + P4 + R848), and VAP (Vaccine + Alum + P4).
Data were obtained using flow-cytometry analysis and CD80 specific antibody. All adjuvant
treated groups contained 4µg of antigen and 1µg each of adjuvant in microparticles.
Figure 4 is a bar graph of mean fluorescence intensity indicating CD86 expression
on DCs following incubation with microparticle formulations for 16 hours. Data were
obtained using flow-cytometry analysis and CD86 specific antibody. All adjuvant treated
groups contained 4µg of antigen and 1µg each of adjuvant in microparticles.
Figure 5 is a bar graph of mean fluorescence intensity indicating CD54 (ICAM-1) expression
on dendritic cells (DC) following incubation with microparticle formulations for 24
hours. Data were obtained using flow-cytometry analysis and CD54 specific antibody.
All adjuvant treated groups contained 7 µg of SP17 antigen and 2 µg of adjuvant in
1 mg of nanoparticle.
Figures 6A and 6B are two bar graphs of mean fluorescence intensity indicating CD40
(left, 6A) and MHC II (right, 6B) expression on DC's following the incubation with
microparticle formulations for 16 hours. Data were obtained using flow-cytometry analysis
and CD40- and MHCII- specific antibodies. All adjuvant treated groups contained 4
µg of antigen and 1 µg of adjuvant in 1 mg of nanoparticle.
Figure 7 is an intensity weighted particle size distribution graph of SP-17 microparticles
obtained using Malvern Zetasizer. SP-17 microparticle size was 3. 59 microns. The
zeta potential was determined to be +9.36mV.
Figure 8 is an image (immunoblot) of sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) gel indicating stability of SP-17 following spray drying. Lanes from right
to left: molecular weight ladder, native SP-17 solution (lane 1), blank microparticles
(lane 2), SP-17 in microparticles (lane 3). Microparticle formulations with or without
SP-17 were suspended in 100 µl PBS (pH 7) and 30 µl was loaded onto the gel. Blank
microparticles were used as a negative control.
Figure 9 is a bar graph of nitrite (stable physiological reservoir of nitric oxide,
in µM) release, which indicates nonspecifically the activation of immune response,
in DCs following the incubation with SP17-loaded microparticles or SP-17 in solution
(or cells without SP17 treatment). Incubation of DCs with SP17 microparticles led
to significant nitrite release compared to DCs incubated with SP17 in solution.
Figure 10 shows that oral vaccination leads to tumor reduction in a breast tumor animal
model. The efficacy and potency of oral nanoparticle vaccine was determined using
the 67NR breast cancer model. Tumor size was measured after oral vaccination with
67NR murine breast cancer cell lysate followed by challenge with live 67NR cells.
Tumor volumes were significantly lower (p<0.01) when compared to the control mice
that received blank particles.
Figures 11A and B show successful uptake of the labeled vaccine particles into the
Peyer's patches of mice. A photomicrograph of distribution of blank nanoparticles
labeled with a fluorescent dye (left, grey dots), and a photomicrograph of a small
intestinal segment (Ileum) in the Peyer's microvillus in the intestine, showing uptake
of particles labeled with IR-CW 800 infrared dye into the Peyer's patches (left image).
Figure 12A and B show localization of oral nanoparticle vaccine to the Peyer's patches.
Rats were administered oral nanoparticle vaccine as described for Figure 11B After
6 hours, bio-images of entire small intestines were taken. Initial segments of intestine
(jejunum, duodenum, items 1 through 6) did not show any presence of dye, while ileum
(items 7 through 12.) showed the presence of dye (shown as white segments). Figure
12C is an enlargement of items 7 and 8 from Figure 12A. Details of Peyer's patches
are visible.
Figure 13 is a plot of percent cumulative release of antigen against the square root
of time from administration, depicting a release profile of antigen from β-cyclodextrin
matrix particles. The best line fit y = f(x) and slope R2 are included. (This figure was published in Chablani et al. J Pharm Sci. 101(10): 3661-71. (2012)).
Figure 14 is a size distribution plot of a batch of microparticles according to the
present disclosure. The microparticles had an average particle size of 1.1 microns
and zeta potential of -31.8 mV. The left ordinate axis represents percentage of particles
passing a given mesh (thereby providing a size cutoff value and the results are plotted
as an S-curve) and the right ordinate axis represents the percentage of volume-based
particle size. The measurements were generated using a Zetatrack Ultra analyzer.
Figure 15 is a microphotograph (taken by scanning electron microscopy) of vaccine
microparticles produced by spray drying. A scale of 5 micron increments is included.
Figure 16A is a plot of mouse body weight over time (with an increase indicative of
implanted ID8 tumor growth) in an ongoing experiment; Figure 16B is a plot of percent
mouse survival over time for each of healthy controls (black squares), mice implanted
with ID8 tumor cells but later vaccinated with microparticles plus SP17 with cytokines
(IL-2 and IL-12, black downward pointing triangles) or with microparticles plus SP17
without cytokines (black diamonds) or with microparticles plus cytokines but without
SP17(black circles) or left untreated (black upward pointing triangles) (n=4 in each
group). After 12 days, survival of the MP + SP17 vaccinated animals even without cytokines
was still indistinguishable from healthy controls whereas all but one mouse in each
control group died.
Figure 17 is a general scheme of vaccine compositions embodiments of the present disclosure.
DETAILED DESCRIPTION
[0038] The present inventors have developed a nanoparticle vaccine formulation for oral
use. In one embodiment, the vaccine composition comprises an aqueous suspension of
β-cyclodextrin (60%), hydroxypropylmethyl cellulose acetate succinate (HPMCAS) nanoparticles,
which carry cancer/testis antigens (CTA), and other molecular species. These are the
microfld (M) cell-targeting Aleuria aurantia lectin (ALL), CpG oligonucleotides or
other adjuvants, and the cytokines, IL2 and IL12. Tumor lysate proteins are also included.
Characteristics of this oral vaccine include a nanoparticle or microparticle formulation
which accesses the host's immune system in Peyer's patches of the gut associated lymphoid
tissue; a targeting of the M cells by a lectin that has not been used with particles
formulation before; a use of cancer testis antigens as the primary immunogen (although
tumor cell lysate antigens can and have been also used); and inclusion of immunostimulatory
molecules (IL-2 and IL-12). The present vaccine composition can be used primarily
therapeutically against various cancers based on the rationale detailed below.
[0039] The present particles assembly and vaccine compositions incorporating such particles
represent the first use of cancer testis antigens (CTA) in an oral vaccine. It had
previously been shown that that CTA are expressed by solid and hematological malignancies,
regardless of the histological origin of the tumor and of the differentiation stage
1-18. For instance, the present inventors and co-workers have previously discovered and
validated a novel CTA panel, including SP17,
1,3,5-9,19-26 Ropporin,
4,5,25AKAP4,
5,10,25,27,28 PTTG1,
25,29,30 which have so far led to the successful pre-clinical validation of these antigens
in different human tumors, namely OC,
3,9,11,31,32 MM,
4,7,8,14,24,28 PC,
10 and LC.
33
[0040] Combining CTA with nanoparticle-based oral vaccine delivery represents an approach
that has never been attempted before and that addresses many of the problems associated
with current vaccine therapies,
such as dendritic cell vaccines, including their high cost. Oral vaccine delivery as proposed is an attractive mode
of immunization because of its ease of administration, low manufacturing cost and
patient acceptability. Generally, orally delivered particulate vaccines are recognized
by microfold (M) cells, which sample the antigen in the Peyer's patches of the intestine
and pass them on to professional antigen presenting cells (APCs) such as dendritic
cells and macrophages that are present in the Peyer's patches
34. These APCs then phagocytose, process and present the particulate antigens on both
MHC Class I and MHC Class II molecules to T-cells. Since dendritic cells have receptors
for both IL-12 and IL-2
5,6,7 and have the capacity to present exogenous antigens to both Class I (cross-presentation)
and Class II pathways
4, the inventors anticipate expect that both T-cell subsets will be shown to be activated
by the
present vaccination methods using the particulate vaccine compositions disclosed here.
Accordingly, the immunostimulatory cytokines IL-2 and IL-12 are also included in some embodiments.
[0041] Encapsulation of proteins into biodegradable polymer particles has been shown to
increase the amount of antigen presented by dendritic cells to the T-cell compartment
by facilitating the endosome escape of encapsulated antigens within the APCs. This
resulted in high efficiency MHC-Class I presentation by APCs and the accompanying
tumoricidal CD8+ CTL response.
35 To enhance the targeting capability of the nanoparticle vaccine formulation to M-cells
in the Peyer's patches of the intestine, the inventors have incorporated the M-cell
targeting agent, Aleuria aurantia (AAL), into vaccine particles. It has recently been
shown that AAL targets nanoparticles to M-cells very efficiently
36. Novel nanoparticle vaccine of the present disclosure also addresses the obstacle
represented by immune suppressor regulatory T-cells (T-reg) in human malignancies.
This innovative vaccine formulation will reduce the T-reg response as indicated by
several lines of evidence using M-cell-targeting vaccines
37. Indeed, the present inventors anticipate that the nanoparticulates are efficiently
taken up by M-cells, and then transferred to mucosal DCs. These are then
anticipated to differentiate towards a CTL-polarizing profile, thus skewing the CTL/Treg balance
38. Although the detailed mechanism has still to be elucidated, it is known that M-cell
targeting vaccines have the potential to attenuate or impair the T-reg response
39, making this strategy highly promising
as an addition to anti-tumor therapies
40. This effect is postulated to be increased by the addition of the TLR-stimulating
CpG or another adjuvant
15. It is also anticipated that addition of IL-2 and IL-12 to the oral nanoparticles
will be shown to induce a more evident antibody response without inducing any toxicity.
In vitro toxicity studies with cultured cells showed no toxicity even at very high
concentrations. General features of the vaccine composition of the present disclosure
is depicted in Figure 17 which is a schematic representation of vaccine emboidmnes
including some of the present disclosure.
[0042] The foregoing nanoparticle formulation has been previously evaluated for oral delivery
of other vaccine antigens such as typhoid antigen
46, tuberculosis antigen
47, prostate cancer vaccine
48, and insulin
49. No toxicity was observed.
[0043] In addition, CTA targeted vaccines have been evaluated for toxicity in animal models
and found safe. See reference 43 below. The CTA vaccine described in this reference
showed no toxicity related to the vaccine in syngeneic mice. No mice exhibited macroscopic
toxicities and post-mortem evaluation of organs (liver, spleen, lungs, kidney, heart,
bone marrow) did not evidence any alteration related to the vaccine. The vaccine consisted
of an injection of full-length recombinant SP17 protein mixed with the CpG adjuvant.
The formulation shares antigen and adjuvant with the one used in experiments herein
but without the use of nanoparticles. Such injection formulation did not produce any
acute or chronic toxicity for the entire duration of the experiments, 300 days, in
vaccinated mice, and therefore the chances of oral nanoparticle-related toxicities
are dramatically low. This is so especially since the doses administered with the
oral microparticulate formulation are much lower (10 times lower for SP17 and 50%
lower for CpG) than those given by injection.
[0044] Moreover, Dr. D'Souza's previously published studies show that high concentrations
of nanoparticulate vaccine are not toxic for macrophages and non-tumoral cells in
vitro. In vitro cyctoxicity assay showed that the nanoparticles are not toxic to the
cells. RAW 264.7 (murine macrophage) cells were incubated with various concentrations
of nanoparticles for 24 hrs and it was found that the nanoparticles were not toxic
up to 2 mg/ml concentration. Furthermore, in another published study by Dr. D'Souza,
where murine breast cancer cells 4T07 were used as a source of antigens for the preparation
of the microparticle vaccine, cytotoxicity studies showed no cytotoxicity at all the
concentrations tested (
Chablani ct al. J Pharm Sci. 101(10): 3661-71. (2012)).
[0045] In another study by the same group involving primates, administration of nanoparticles
(without CTA) did not affect any normal physiological parameters. No allergic reactions
or antibodies against the formulation were detected
3. These results show that our proprietary microparticulate formulation is non-toxic
and highly safe even in large animals (primates). Therefore, toxicities of the present
vaccine compositions in vivo are unlikely.
Particles and Preparation
[0046] As used herein, the terms "microparticles" and "nanoparticles" are used interchangeably
in the sense that what is said about one can also apply to the other. The term "particles"
is used to denote microparticles or nanoparticles, as those term are used in the art,
or mixtures thereof. Typically, microparticles have an average (mean) diameter range
beween about 1 and about 5 microns.
[0047] Particles and therefore particle assemblies in accordance with the present disclosure
can be made by spray drying methods, preferably in one step, by mixing the various
matrix and active ingredients, in an aqueous medium spraying in droplets of controlled
size and drying the droplets to form microparticles or nanoparticles or both. Detailed
methods and ingredients for making microparticles in accordance with the invention
is disclosed in PCT Patent Application
PCT/US2009/058896 published as
WO 2010/037142. See the entire disclosure, (including the claims) which is incorporated by reference
in its entirety and especially see pp. 48 to 56. Physical characteristics of the particles
(such as size morphology, distribution and zeta potential and any other characteristic
assessed in the pharmaceutical API or formulation particle art) can be assessed by
any known method, for example by methods described below in the Examples. In some
embodiments, the ingredients for the particle matrix include a water soluble polymer
such as beta cyclodextrin and enteric coating materials such as ethuyl cellulose (EC)
and/or hydroxypropylmethyl cellulose acetate succinate (HPMCAS). In some embodiments,
the matrix ingredients form approximately 80 to 95% of the particles, with the active
ingredients forming approximately 5 to 20%.
[0048] In some embodiments, approximate relative amount ranges (w/w) of the matrix ingredients
based on the weight of the particles are as follows:
Beta-cyclodextrin: 40 to 80 %;
Combined EC and HPMCAS: 10 to 35 % .
[0049] For example, in some embodiments, the approximate amount of ethyl cellulose is from
0 to 15 % and that of HPMCAS is 10 to 35 %.
[0050] More details on the approximate relative amounts of the active ingredients (cancer
testis antigens(CTA), lectin, cytokine, adjuvant and tumor lysate proteins) follow
in each section. Of these, each one of cytokine, adjuvant and lysate and combinations
of two or all three of them are preferably included as will be illustrated below.
[0051] In a particular formulation employed in the present experiments the matrix contains
approximately 60% beta cyclodextrin, 20% HPMCAS, 15% EC. The remainder is CTA. The
optional ingredients are added to this formulation in percentages within the ranges
disclosed herein.
[0052] The nanoparticles having a particular formulation of 60% beta cyclodextrin, 30% HMPCAS,
15% EC used in the experiments described in the examples have been evaluated
Cancet Testis Antigens (CTA)
[0053] The present disclosure features the use for the first time in an oral vaccine of
one or more CTA. CTA have been identified as being expressed in a variety of tumors
as can be seen from many of the cited references at the ed of this specification.
If additional CTAs are identified in the future as expressed (especially overexpressed)
in both solid and hematologic tumors such CTAs will be suitable for use in the present
formulations and methods.At least one CTA can be used hence combinations of more than
one CTA are within the scope of the disclosure. Of course the more overexpressed is
a CTA in a particular tumor, the more attractive it is as an immunogen.
[0054] CTAs already identified as potential therapeutic targets include SP17, AKAP4, PTTG1,
and Ropporinl, ASP, MAGE, NY-ESO-1, HER2neu, and Hm1.24 and combinations of two or
more of the foregoing. CTAs that are expressed and have been identified as targets
in tumors are disclosed in several of the references that follow which are incorporated
by reference in their entirety (for example Nos 13, 15, and 24). in the references
listed at the end of the present specification. MAGE group members that are used in
some embodiments include without limitation MAGE-A3, MAGE-A9 and MAGE-A12.
Lectins
[0055] Lectins that may be used in the methods, vaccine compositions and particles of the
present disclosure include without limitation one or more lectins that target microfold
cells in Peyer's patches. Examples are wheat germ agglutinin (WGA), Ulex europaues
1 (UEA-1), Concavalin A (ConA) and Aluria aurantia lectin (AAL) which in certain embodiments
is preferred. Even if a lectin does not target a particular vaccine to the M-cells
of a particular subject effectively, this does not mean that it will perform satisfactorily
with a different vaccine within the present disclosure in a different subject or using
a different antigen and/or batch of lectin as long as the lectin has been shown to
target M cells in at least one experiment.
[0056] The amount of lectin or lectins that is typically used in the present particles,
vaccine compositions and methods is within the range of about 0.1 to about 0.5% (w/w)
based on the weight of the particles.
Adjuvants
[0057] Adjuvants suitable for use in the methods, compositions and particles of the present
disclosure include without limitation any pharmaceutically acceptable adjuvant that
will stimulate the initial innate immunity response to antigen (CTA and tumor lysate
protein if the latter is used) and thus potentiate immunogenicity of a CTA given by
oral route. Nonlimiting examples include CpG, MF59, alum, flagellin, R848, monophosphoryl
lipid A, ODN 1826, and combinations of at least two of the foregoing. Even if an adjuvant
does not potentiate the immune response with a particular vaccine and a particular
CTA, as long as it has been demonstrated to have adjuvant activity in at least one
system, that adjuvant is potentially useful in another embodiment of the present disclosure.
[0058] In the Examples below, CpG was used as adjuvant when an adjuvant was used as it has
been been shown to provide signals for dendritic cells and T-cell activation
41,42 and has been shown to provide an efficient adjuvant effect in oral vaccines formulations
45. However, the remaining adjuvants listed above were also screened. The rationale
for using one or more of them instead or in addition to CpG is as follows:
[0059] In place of CpG, several different adjuvants can be used in the same nanoparticulate
formulation, to stimulate the initial innate immunity response and thus to ameliorate
the vaccine efficacy. The activation of the immune response can be maximized by designing
the molecular topology of the microparticle according to so-called pathogen-associated
molecular patterns (PAMPs). These patterns represent small molecular sequences, which
are consistently found on pathogens. Adjuvants mimic these PAMPs, thus improving the
response to nanoparticle vaccines
in vivo and
in vitro.
[0060] The present inventors have screened the following adjuvants as potentiators of the
present particles and vaccine compositions:
[0061] AddaVax™(MF59) is a squalene-based oil-in-water nano-emulsion based on the formulation
of MF59 that has been licensed in Europe for adjuvanted flu vaccines. Squalene is
an oil more readily metabolized than the paraffin oil used in Freund's adjuvants.
AddaVax™ promotes a significant increase in antibody titers with reportedly more balanced
Thl/Th2 responses than those obtained with alum. MF59is available from Novartis AG.
AS03 (Adjuvant System 03) is another squalene based adjuvant commercially available
from Glaxo SmithKline. A dose of AS03 adjuvant contains 10.69 mg squalene 11.86 mg
DL-α-tocopherol .86 mg polysorbate 80.
[0062] Alhydrogel 2% is an aluminium hydroxide (referred to as alum) wet gel suspension.
Alum improves attraction and uptake of antigen by APCs. It has been suggested that
the antigens absorbed on the aluminum salts are presented in a particulate form, making
them more efficiently internalized by APCs. Alum increases Th2 response (leading to
antibody production) but does not promote significant Th1 cellular response.
[0063] Flagellin (FliC) is a recombinant flagellin protein encoded by the fliC gene from
Salmonella typhimurium. Unlike other TLR agonists, flagellin tends to produce mixed
Th1 and Th2 responses rather than strongly Th1 responses. It has been demonstrated
that flagellin can act as a potent adjuvant in flu vaccines.
[0064] R848 is imidazoquinoline compounds and agonists for TLR7 and TLR8. They are effective
adjuvants by activating dendritic cells (DCs) and B cells to induce cytokines optimal
for Th1 cell immunity, and antibody production.
[0065] MPLA (monophosphoryl lipid A), a TLR4 agonist, is a derivative of lipid A from Salmonella
minnesota R595 lipopolysaccharide (LPS or endotoxin). MPLA is considerably less toxic
than LPS whilst maintaining the immunostimulatory activity. When tested in animals
models as a vaccine adjuvant, MPLA induces a strong Th1 response.
[0066] ODN 1826 are synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG
ODNs). CpG ODNs are recognized by TLR9, which is expressed exclusively on human B
cells and plasmacytoid dendritic cells (pDCs), thereby inducing Th1-dominated immune
responses. Pre-clinical and clinical trials have demonstrated that CpG ODNs can significantly
improve vaccine-specific antibody responses. ODN1826 is commercially available, e.g.,
fromInvivoGen, San Diego CA 92121 (website domain: invivogen.com)
[0067] P4 is a 28-30 kDa lipoprotein present in all typeable and nontypeable strains of
H. influenzae. P4 is an attractive adjuvant; however, the ability of P4 to induce antiboides protective
against infections is still controversial. One P4 isolate has the deduced amino acid
sequence GenBank accession no. P26093.
[0068] The amount of the at least one adjuvant that is typically used in the particles,
vaccine compositions and methods of the present disclosure is within the range from
about 0.1 to about 0.5 % (w/w) based on the weight of the particles.
Cytokines
[0069] Cytokines such as IL-2 and IL-12 which have been shown to provide signals for dendritic
cells and T-cell activation, and adjuvants such as CpG which has been shown to enhance
the efficacy of weak tumor antigens and promote T cell responses
42,43, are advantageously included in the present CTA-containing oral vaccine compositions,
particles and methods. As the present inventors and their co-workers have recently
described with oral vaccines using lysates of the murine ovarian cancer ID8 cells
as autologous (without additional CTAs) targets
44.
Tumor Cell Lysate Antigens
[0070] The immune response to cancer is largely determined by the way in which tumor cells
die. As necrotic, stress-associated death can be associated with activation of antitumor
immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based
vaccination have commonly used "necrotic" lysates as an immunogenic source of tumor-associated
antigens. Thus, due to the presence of many relevant immunogenic epitopes, whole tumor
lysates are promising antigen sources for dendritic cell (DC) therapy, as such immunogenic
epitopes can help prevent tumor escape.
[0071] In one embodiment, the compositions of the present disclosure comprise one or more
tumor-associated antigens, where tumor-associated antigens can be provided by a tumor
cell lysate. Preferably, an autologous tumor cell lysate is provided, i.e. a lysate
from a tumor derived from the patient to be treated. However, it is also possible
to use tumor-associated antigens from a tumor cell line. In that event, preferably,
a tumor cell line of the same tumor type as the tumor to be treated is used.
[0072] In another embodiment, both autologous and allogeneic tumor cell lysates can be used
for the vaccine formulations of the present disclosure. For example, a tumor vaccine
composed of three irradiated allogeneic melanoma cell lines demonstrated promising
results in a phase II clinical trial but was terminated in a phase III study due to
low efficacy (
Drake et al. Nat. Rev. Clin. Oncol. 11, 24-37 (2014). Thus, the vaccine formulations of the present disclosure may encompass one or more
tumor cell lysates, which can be either autologous or allogeneic.
[0073] Any method suitable for lysate preparation can be used in preparation of vaccine
microparticles of the present disclosure. Two common methods of whole tumor cell lysate
preparation are ultraviolet B (UVB) ray-irradiation and repeat cycles of freezing
and thawing. More recently, hypochlorous acid (HOCl)-oxidation method has also been
used for inducing primary necrosis and enhancing the immunogenicity of tumor cells.
Since vaccine efficacy can vary depending on the method of lysate preparation used,
vaccine formulations containing lysates prepared by different lysis protocols (freeze-thaw,
UVB, or HOCI) can be assessed in order to determine which protocol provides optimal
vaccine efficacy.
[0074] While variations can be introduced into each cell lysis protocol, as is appreciated
by and known to those skilled in the art, the following exemplary protocols illustrate
general features of lysis procedures.
[0075] Freeze-thaw method: The confluent cells are washed with cold phosphate buffered saline
(PBS). The flasks are then treated with hypotonic buffer (10mM Tris and 10mM NaCl)
and subjected to five 15 min freeze-thaw cycles at temperatures of -80 °C and 37°
C respectively to obtain the cell lysate. This protocol was used in the experiments
described in the Examples below when tumor lysate was used.
[0076] UVB irradiation method: For UVB-irradiation, confluent cells are subjected to a UVB-irradiation
for 10 min to induce apoptosis. Cells are then incubated overnight at 37C, 5% CO2,
and harvested on the following day for 5 cycles of freeze-thaw treatment (for example,
freezing with dry ice for 20 min and thawing at room temperature) before use.
[0077] HOCI oxidation method: HOCl solution is prepared by diluting the stock sodium hypochlorite
solution (NaOCl, reagent grade, available chlorine 10-15%; Sigma-Aldrich Corp.) with
DPBS (Cellgro) and added immediately to tumor cells. The tumor cell suspension is
then incubated for 1 h at 37°C, 5% CO2 with gentle agitation after every 30 min to
induce oxidation-dependent tumor cell death. After that, tumor cells are harvested,
washed twice with DPBS and resuspended at 1 × 107 cells/ml in appropriate DC media
for 6 cycles of freeze (either with dry ice for ≥ 20 min or at -80°C for ≥ 1 h) and
thaw at room temperature to complete fragmentation before loading onto DCs.
Tumor Types
[0079] In one embodiment, the present disclosure relates to methods for treating a patient
afflicted with a malignant tumor. In some embodiments, the tumor is a solid tumor.
In other embodiments, the tumor is a hematologic tumor.
[0080] In some embodiments, the tumor is selected from the group consisting of adrenal (e.g.,
adrenocortical carcinoma), anal, bile duct, bladder, bone (e.g., Ewing's sarcoma,
osteosarcoma, malignant fibrous histiocytoma), brain/CNS (e.g., astrocytoma, glioma,
glioblastoma, childhood tumors, such as atypical teratoid/rhabdoid tumor, germ cell
tumor, embryonal tumor, ependymoma), breast (including without limitation ductal carcinoma
in situ, carcinoma, cervical, colon/rectum, endometrial, esophageal, eye (e.g., melanoma,
retinoblastoma), gallbladder, gastrointestinal, kidney (e.g., renal cell, Wilms' tumor),
heart, head and neck, laryngeal and hypopharyngeal, liver, lung, oral (e.g., lip,
mouth, salivary gland) mesothelioma, nasopharyngeal, neuroblastoma, ovarian, pancreatic,
peritoneal, pituitary, prostate, retinoblastoma, rhabdomyosarcoma, salivary gland,
sarcoma (e.g., Kaposi's sarcoma), skin (e.g., squamous cell carcinoma, basal cell
carcinoma, melanoma), small intestine, stomach, soft tissue sarcoma (such as fibrosarcoma),
rhabdomyosarcoma, testicular, thymus, thyroid, parathyroid, uterine (including without
limitation endometrial, fallopian tube), and vaginal tumor and the metastasis thereof.
In some embodiments, the tumor is selected from the group consisting of breast, lung,
GI tract, skin, and soft tissue tumors. In some further embodiments the tumor is selected
from the group consisting of breast, lung, GI tract and prostate tumors.
[0081] In some embodiments, the tumor is selected from the group consisting of leukemia,
lymphoma, and myeloma. In some further embodiments, the tumor is selected from the
group consisting of myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic
myelogenous leukemia (CML), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic
lymphocytic leukemia (CLL), and multiple myeloma.
Vaccine Unit Dosage and Administration Including Booster Schedule
[0082] In one embodiment, the dose of the oral vaccine for the use in mice is in the range
of 50 µg -5 mg /dose/mouse, where the upper limit dose of 5 mg/dose/mouse is based
the ongoing animal experiments and then may end-up being higher or lower within the
same order of magnitude. In another embodiment, the dose of the oral vaccine for the
use in mice is in the range of 100 µg - 4 mg/dose/mouse. In a more specific embodiment,
the dose of the oral vaccine for the use in mice is in the range of 500 µg - 2.5 mg/dose/mouse.
[0083] In some embodiments, the vaccine is administered as one prime dose followed by several
booster doses. The number of booster doses is in the range of 1-10 booster doses.
In one embodiment, the vaccine is administered as one prime followed by 2-8 booster
doses. In another embodiment, the vaccine is administered as one prime followed by
3-7 booster doses. In a more specific embodiment, the vaccine is administered as one
prime followed by 6 booster doses.
[0084] In one embodiment, booster doses are administered at intervals within the range fom
about every 5 days to about 3 weeks. In another embodiment, a booster dose is administered
every 2 weeks or even weekly.
[0085] In one embodiment, the dosage of adjuvant administered to mice is in the range of
2-30 µg/dose/mouse. In another embodiment, the dosage of adjuvant is in the range
of 5-25 µg/dose/mouse. In a further embodiment, the dosage of adjuvant is in the range
of 7.5-20 µg/dose/mouse.
[0086] In one embodiment, the dose of the oral vaccine for the use in human subjects is
in the range of 5mg to 320 mg. In one embodiment, the oral vaccine is administered
at an initial dose of 5 mg to a human subject, after which the dosage is escalated
to 320 mg per dose (320 mg per dose will be the final dosage in the phase I safety
trial). It is anticipated that depending on the results obtained during the phase
I study, the dosage requirements may change.
[0087] In another embodiment, the dose of the oral vaccine for the use in human subjects
is in the range of 10mg to 250 mg. In further embodiment, the dose of the oral vaccine
for the use in human subjects is in the range of 20 mg-200 mg.
[0088] In one embodiment, the oral vaccine of the present disclosure is administered to
a human subject as one prime dose followed by several booster doses. In one embodiment,
the number of booster doses varies in the range between 1-10 booster doses during
the course of treatment. In one embodiment, the vaccine is administered as one prime
followed by 2-8 booster doses. In another embodiment, the vaccine is administered
as one prime followed by 3-7 booster doses. In a more specific embodiment, the vaccine
is administered as one prime and 6 booster doses.
In one embodiment, one or more booster doses are administered at intervals within
the range of 1-3 weeks. In another embodiment, a booster dose is administered every
10 to 18 days. In a preffered embodiment, a booster dose is administered every 2 weeks.
[0089] In one embodiment, the dosage of adjuvant administered to a human subject is in the
range of 50-500 ug/dose/human. In another embodiment, the dosage of adjuvant administered
to a human subject is in the range of 100-400 ug/dose/human. In a further embodiment,
the dosage of adjuvant administered to a human subject is in the range of 100-400ug/dose/human.
EXAMPLES
Example 1
Preparation of Vaccine Nanoparticles
[0090] The inventors have developed a biodegradable and biocompatible polymer matrix system
for oral vaccines composed of β-cyclodextrin (60%), hydroxypropylmethylcellulose acetate
succinate (HPMCAS, 20%), and ethyl cellulose (EC, 15%). The HPMCAS was incorporated
because of its enteric properties to protect the proteins from the harsh gastric conditions.
By the way of general description of the particles, the β-cyclodextrin and ethyl cellulose
function as the sustained release polymer matrix. These compounds are regarded as
safe (GRAS) by the FDA for human use. The oral vaccine formulation contained one or
more cancer testis antigens (CTA) or miscellaneous tumor-derived antigens prepared
from whole tumor cell lysates. Furthermore, the vaccine formulation also contained
microfold (M)-cell targeting ligand Aleuria Aurantia lectin (AAL, 0.25% w/w loading),
which has been shown to improve the targeting of the particles to the Peyer's patches.
Generally, M cells act as sampling ports for any foreign molecules encountered in
the small intestine, and have been shown to house numerous dendritic cells and immune
cells. Following the sampling of the oral vaccine by M cells, the particle is processed
by a dendritic/antigen presenting cell (APC) and presented on major histocompatibility
complex (MHC) class I or MHC class II molecules. CpG oligodeoxynucleotides, which
have been shown to provide signals for the dendritic cell and T-cell activation were
used as an adjuvant in the formulation (
Emtage et al. J Interferon Cytokine Res. 18(11): 927-937 (1998)). CpG has also been shown to enhance the efficacy of weak tumor antigens and to
promote T cell responses (
Zhang et al. J Immunother.30(5):469-478 (2007),
Chiriva-Internati et al. PLoS One. 5(5):e10471 (2010)). Furthermore, the vaccine formulations described herein also contained immunostimulatory
cytokines IL-2 and IL-12, which have been shown to provide signals for dendritic cell
and T- cell activation (
Tawde et al. Vaccine. 30(38):5675-5681 (2012)). Finally, the vaccine polymer solution was spray-dryed through Buchi 191 Spray
Dryer. The spray drying procedure aerosolizes the vaccine antigen-polymer matrix,
where optimum water removal from the droplet results in nanoparticles containing the
vaccine antigen in a polymer matrix (schematically represented in Fig. 1)).
[0091] Vaccine particles described herein were formulated using the ID8 cell lysate or SP17
protein (5%, w/w), β-cyclodextrin, ethyl cellulose, human or mouse plasma, CpG, HPMCAS,
and targeting agent AAL dissolved in deionized water. Briefly, HPMCAS were dissolved
in an alkaline solution. Human or mouse plasma was added to the polymeric solution
at pH 7.0, which contains albumin (although albumin was not part of the basic particle
system). AAL was added to the solution, followed by the addition of ID8 lysate or
SP17 protein (5%, w/w). The CpG sequence #1826 was also included in this mixture.
The weight ratio of CpG: nanoparticle polymer was 1:500. This corresponds to a dose
of 10 µg CpG per dose of nanoparticle vaccine (5 mg/mouse), which has been previously
shown to provide an efficient adjuvant effect in oral vaccines formulations (
McCluskie et al. Vaccine. 19(4-5):413-422 (2000)). Finally, this aqueous solution was spray dried by one-step process using the Buchi
B-191 mini spray dryer (Buchi Corporation, New Castle, Delaware) at inlet temperature
125° C, outlet temperature 80° C, 500 L/h, and 2% feed rate (20 mL/h) of peristaltic
pump, and nozzle diameter of 0.7 mm (
Akande at al. J Microencapsul.27(4):325-336 (2010)). The resulting particles can be stored at -20°C in a desiccant chamber till further
use.
Example 2
Testing of Various Adjuvants as Potentiators of SP17 Nanoparticle Vaccine
[0092] In place or in addition to CpG, several different adjuvants can be used in the same
particulate formulation. Inclusion of additional adjuvants can further stimulate the
initial innate immunity response and thus ameliorate the vaccine efficacy. The activation
of the immune response can be maximized by designing the molecular topology of the
microparticle according to pathogen-associated molecular patterns (PAMPs). These patterns
represent small molecular sequences, which are consistently found on pathogens (
Mogensen TH, Clin Microbiol Rev. 22(2): 240-273 (2009)). Adjuvants mimic these PAMPs, thus improving the response to nanoparticle vaccines
both
in vivo and
in vitro.
[0093] The following adjuvants have been screened as potentiators of nanoparticle oral vaccine
formulation described in the present invention:
∘ AddaVax™(MF59) a squalene-based oil-in-water nano-emulsion
∘ Alhydrogel 2% an aluminium hydroxide adjuvant (referred herein to as alum) available
as wet gel suspension..
∘ Flagellin (FliC) a recombinant flagellin protein encoded by the fliC gene from Salmonella
typhimurium.
∘ R848 an imidazoquinoline compound and agonist for TLR7 and TLR8.
∘ MPLA (monophosphoryl lipid A), a TLR4 agonist that is a derivative of lipid A from
Salmonella minnesota R595 lipopolysaccharide (LPS or endotoxin).
∘ ODN 1826 a group of synthetic oligodeoxynucleotides containing unmethylated CpG
motifs (CpG ODNs).
∘ P4 a 28-30 kDa lipoprotein present in all typeable and nontypeable strains of H. influenzae.
[0094] For the screening of various adjuvants (described above 1-7, Table 1), nanoparticles
were prepared as described above (Example 1). Cancer/testis antigen (CTA) SP17 was
used as a target antigen. Briefly, 400,000 human monocyte-derived dendritic cells
(DC) were incubated with 0.2 mg of vaccine nanoparticles (5 µg total protein) and
0.2 mg adjuvant nanoparticles (2 µg adjuvant). Dendritic cells were incubated with
the nanoparticles for 24 hours. Following the incubation period, cells were washed
with PBS and incubated with specific markers for flow-cytometry analysis. All adjuvant
treated groups included 7 µg of SP17 in macroparticles (MP) and 2 µg of antigen in
MP.
Table 1. Vaccine Formulations Containing Various Adjuvants
Formulation |
Formulation Content |
VM |
Vaccine + MF59 |
VP |
Vaccine + P4 |
VR |
Vaccine + R848 |
VMR |
Vaccine+ MF59 + R848 |
VAM |
Vaccine + Alum + MF59 |
VMP |
Vaccine + MF59 + P4 |
VPR |
Vaccine + P4 + R848 |
VAP |
Vaccine + Alum + P4 |
[0095] Nitric oxide is a non-specific marker of immune system activation. Concentrations
of nitrite (NO
2-) levels reflecting nitric oxide production in DCs can be measured with the Griess
reagent system (Promega). As shown in Figure 2, most of the adjuvant treated groups
showed a statistically significant (p< 0.05) increase in nitric oxide levels compared
to the SP17 (SP) microparticles (MP) alone. Flagellin and MPLA were the only adjuvants
that did not cause an increase in nitric oxide levels compared to SP17 MP alone. However,
while Flagellin and MPLA inclusion did not cause an increase in nitric oxide levels
in this specific example, it cannot be said that these adjuvants would not lead to
immune system activation if tested using a different system (host, . Thus, the inventors
anticipate that t inclusion of Flagellin and/or MPLA into the vaccine formulations
of the present disclosure even in the absence of other adjuvants can lead to enhanced
activation of the immune system. Furthermore, Flagellin and MPLA may contribute to
the specific immune response such as upregulation of DC markers (see Example 4).
Example 3
Addition of Adjuvants to SP17 Nanoparticle Vaccine Enhances the Activation of Dendritic
Cells
[0096] Signaling via CD80 and CD86 regulates dendritic cell activation and maturation. Given
the results described in Example 2 and the observation that most of the formulations
containing adjuvants led to nitric oxide release in DCs, the inventors sought to further
evaluate the immune response following the incubation of DCs with microparticle formulations
described in Table 1. Briefly, DCs were treated with microparticle formulations for
16 hours, where each group treated with adjuvants received 4 µg of antigen and 1 µg
of the corresponding adjuvant(s) in microparticles. Following the 16-hour incubation
period, cells were washed with PBS and evaluated for CD80 and CD86 marker expression
using flow-cytometry analysis. As shown in Figures 3 and 4, both CD80 (Figure 3) and
CD86 (Figure 4) expression was increased following the incubation of DCs with vaccine
microparticles containing an adjuvant, compared to the vaccine alone. Furthermore,
DCs exposed to formulations containing more than one adjuvant (VPR, VPM, and VMR)
exhibited even higher expression of CD80 and CD86 compared to DCs treated with vaccine
microparticles with a single adjuvant.
[0097] These experiments show that: (1) treatment of cells with vaccine microparticles along
with an adjuvant results in the increased activation of DCs; and (2) inclusion of
additional adjuvants (more than one) into the formulation further intensifies DC activation.
Example 4
Inclusion of an Adjuvant into Microparticle Formulations Leads to the Induction of
ICAM-1 expression in DCs
[0098] Adhesion receptors such as Intracellular Adhesion Molecule (ICAM-1) (also known as
CD54) are expressed on mature dendritic cells and function to promote adhesion to
T cells. Next, the inventors tested the ability of DCs treated with SP17 microparticle
formulations containing different adjuvants to upregulate ICAM-1 expression. Briefly,
DC 2.4 cells were incubated with and without an adjuvant. All DCs treated with an
adjuvant received 7 µg SP17 antigen and 2 µg of adjuvant in 1 mg of nanoparticle.
As demonstrated in Figure 5, ICAM-1 expression was elevated in MPLA and P4 treated
groups (p<0.001) compared to SP17 MP.
[0099] Thus, in addition to the increase in CD80 and CD86 expression, inclusion of adjuvants
in the microparticle formulations also increases ICAM-1 expression, which reflects
conditions of DC maturation (the expression of ICAM-1 is low in immature DCs and increases
upon maturation).
Example 5
Addition of Adjuvants to SP17 Nanoparticle Vaccine Leads to Increased CD40 and MHC
II Expression in DCs
[0100] Next, expression of CD40 and MHC II were analyzed as stimulation markers of DCs.
DCs were incubated with various microparticle formulations (Table 1) for 16 hours.
All adjuvant-treated groups received 4 µg of antigen and 1 µg of adjuvant in 1 mg
of nanoparticle. As shown in Figure 6, the pattern of CD40 and MHC II expression is
similar to that observed for CD80 and CD86 (Example 3), where it was shown that CD80
and CD86 expression on DCs increases with the inclusion of additional adjuvants as
compared to vaccine mp with a single adjuvant. Figure 6 demonstrates that CD40 and
MHC II expression was elevated in cells receiving vaccine microparticle along with
combination of adjuvants (p<0.05) compared to vaccine MP with a single adjuvant.
Example 6
Characterization of Physical and Biological Properties of Nanoparticles
[0101] The inventors analyzed the physical properties of the nanoparticles used in the foregoing
experiments. The particle size, and zeta potential was determined using a Malvern
Zeta Sizer. Initial studies focused on SP-17 loaded nanoparticles. Prototype studies
produced batches with consistent size ranges of around 3.59 microns, which was in
the desired particle size range of 1-5 microns (Fig. 7) and the zeta potential was
around + 9.36 mV. Furthermore, scanning electron microscopy revealed that the particles
are almost spherical but they have a rough surface.
[0102] Furthermore, the stability of the vaccine antigen upon encapsulation (following spray
drying process) was evaluated in order to determine whether spray drying leads to
degradation and/or instability of the vaccine antigen. Sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) analysis showed that the vaccine antigen remains stable
on spray drying (Figure 8).
[0103] Most successful vaccines are generated by activation of dendritic cells via multiple
Toll-like receptors (TLRs) to stimulate pro-inflammatory cytokines. The triggering
of combinations of TLRs can induce synergistic production of cytokines leading to
the enhanced T-cell response. The inventors next evaluated the immune response in
terms of the release of non-specific markers like nitric oxide (Figure 9). Dendritic
cells were prepared from human peripheral blood monocytes as previously described
(
Chiriva-Internati et al. PLoS One. 5(5):e10471 (2010);
Stagg et al. Methods Mol Med. 64:9-22 (2001)). DCs were plated in a 96-well plate and after 2 hours SP17 antigen loaded nanoparticles
were added to the cells. Blank nanoparticles and antigen in solution were used as
controls. Nanoparticles or solution were allowed to be in contact with the dendritic
cells for a period of 48 hours. After the 48-hour incubation period, the cell supernatant
was removed and analyzed for nitrite (readout for nitric oxide) release (Fig. 9).
As shown in Figure 9, incubation of human monocytes with SP17 microparticles led to
a significant release of nitric oxide compared to the SP17 in solution. These findings
indicate the activation of the innate immune response via treatment with SP17 antigen
loaded particles in human dendritic cells.
Example 7
In Vivo Oral Vaccine Studies using Breast Cancer Animal Model
[0105] In this Example, the inventors conducted oral nanoparticle vaccine efficacy and potency
determination studies using the 67NR breast cancer model. Animals were primed with
5 mg of either blank or vaccine microparticle (equivalent to 250 µg of 67NR antigen)
suspension in 200 µL of citrate buffer (10 mM, pH 4.0) using oral feeding needle.
One week after the initial dose, two booster doses of same strength were given in
alternate weeks.
[0106] After the final booster animals were challenged with IX 106 live murine 67NR tumor
cells intra-peritoneally to determine the efficacy of 67NR microparticulate vaccine.
Briefly, 67NR tumor cells were suspended in 100 µL of serum-free DMEM medium and injected
subcutaneously into animals. Following 2 weeks (post-injection of tumor cells), vaccination
resulted in significantly lower tumor volumes compared to the control (Fig 10).
[0107] In order to gain insight into the uptake of vaccine particles into Peyer's patches
of animals following oral administration, the inventors conducted vaccine uptake studies
using labeled biopolymer nanoparticles. Use of nanoparticles labeled with fluorescent
dyes crystal violet (Figure 11A, left) and IR dye CW-800 (Figure 11B, right)) indicated
excellent uptake of particles into the Peyer's patches. Furthermore, Figure 12 shows
that oral nanoparticle vaccine described herein localized to the Peyer's patches.
[0108] Regarding the stability of the antigen-nanoparticle complexes, it is anticipated
that 67NR tumor antigen-nanoparticle complexes exhibit stability qualitatively similar
to that observed for another breast cancer antigen, 4T07 (Figure 13 taken from
Chablani et al. J Pharm Sci. 101(10): 3661-71. (2012)). There, in the case of 4T07 antigen, dissolution studies demonstrated sustained
release of antigen from the microparticles.
Example 8
Preparation of bio-degradable vaccine nanoparticles containing ovarian cancer cells
lysate
[0109] The inventors prepared a bio-degradable vaccine nanoparticles containing ID8 ovarian
cancer cell lysate (specifically lysate proteins or antigens) in addition to SP17
antigen.
Preparation of tumor lysate and SP17 mouse protein:
[0110] The ID8 ovarian cancer cells were cultured for 3 days in 75 cm
2 tissue culture flasks in a 5% CO
2, 37°C incubator until sub-confluent. The cells were washed with Phosphate Buffered
Saline (PBS) pH 7.4 and scraped in the presence of hypotonic buffer. Subsequently,
the cells were subjected to five freeze-thaw cycles, which includes freezing the cell
suspension at - 80 °C for 10 minutes followed by thawing at 37 °C degrees for 10 minutes.
The lysate was stored at -80 °C and used to prepare the vaccine. This is a standard
protocol for the preparation of cell lysate and can be used to prepare cell lysates
of various different cell types.
[0111] His-tagged recombinant SP17 mouse protein was expressed in
E. coli and purified in the laboratory following standard protocols.
Preparation of the vaccine nanoparticles:
[0112] The matrix system used for the microparticles of this oral vaccine composition contained
β-cyclodextrin (60%), hydroxy propyl methyl cellulose acetate succinate (HPMCAS, 20%)
and ethyl cellulose (EC, 15%). Briefly, the biopolymers were dissolved in water together
with the SP17 vaccine antigen (5% w/w). The vaccine formulation also contained antigens
derived from the ID8 ovarian cancer cell lysate (10%), as well as M-cell targeting
ligand Aleuria Aurantia lectin (AAL), that has been shown to improve the targeting
of the particles to the Peyer's patches. In addition, immunostimulatory cytokines
(IL-2 and IL-12), and CpG oligodeoxynucleotide were also included in the vaccine formulation.
The vaccine formulation also contained mouse plasma. The vaccine polymer solution
was spray dried through a Buchi B191 Spray Dryer (Buchi Corporation) and optimum water
removal from the droplets resulted in nanoparticles containing the vaccine antigen
and other components in a polymer matrix.
Example 9
Characterization of the ID8 and SP17 Antigen Containing Vaccine Nanoparticles
[0113] The particle size and zeta potential were determined using a Zetatrac Ultra zeta
potential and particle size analyzer (Figure 14). Average particle size of polymer
microparticles was found to be 1.1 microns. The zeta potential of the microparticles
was determined to be -31.8 mV. The antigen content after spray drying was assessed
by SDS-PAGE analysis, which confirmed a stable SP17 content following spray drying.
The particle vaccine formulation was highly stable with a maximum 60% release (data
not shown). Furthermore, the morphology of the spray dried particles was determined
by scanning electron microscopy (Figure 15).
[0114] The vaccine preparation was next evaluated to determine the release kinetics of the
encapsulated material. The particulate vaccine formulations were suspended in simulated
gastric fluid for 2 hours followed by intestinal fluid with the goal to mimic the
conditions of the stomach and intestine. The vaccine antigen content wasdetermined
by a micro BCA protein assay method (commercially available, Thermo Fisher Scientific
Inc, Waltham, MA USA 02451). The nanoparticle vaccine formulation was highly stable,
with no more than 55% of antigen released after 5 hours.
Example 10
In Vitro Biological Evaluation of the ID8 and SP17 Antigen Containing Vaccine Nanoparticles
[0115] Next, the best vaccine formulation was selected by taking the advantage of evaluating
the innate immune response
in vitro in dendritic cells. Thus, the immune response was measured in terms of the release
of the non-specific marker nitric oxide by DCs. As a negative control, the inventors
used either blank nanoparticles or antigen in solution. The nanoparticles or antigen
solution were incubated with DCs for a period of 48 hours. After the incubation period,
the cell supernatant was removed and analyzed for nitric oxide release. Incubation
of DCs with SP17 microparticles showed a significant increase in nitric oxide release,
indicating the activation of the innate immune system.
Example 11
In Vivo Biological Evaluation of the ID8 and SP17 Antigen Containing Vaccine Nanoparticles
[0116] C57/B16 mice (N=16) were injected with 2 x 10
6 ID8 cells intra-peritoneally. Control mice (N=4) were injected with PBS. Animal weight
and signs of ascites were recorded every other day. After 37 days, mice with comparable
tumors were divided into 4 groups (N=4/group) and the oral vaccination schedule was
initiated (Table 2). Animals were boosted with the vaccine 3 times, where each treatment
followed the prior one by one week.
Group |
Treatment |
1 |
0.2 mL citrate buffer (untreated tumor) |
2 |
5 mg MP + cytokines+SP17 (full microparticle formulation) in 0.2 mL citrate buffer |
3 |
5 mg MP + cytokines (microparticles without SP17) in 0.2 mL citrate buffer |
4 |
5 mg MP + SP17 (microparticles without cytokines) in 0.2 mL citrate buffer |
[0117] While these studies are ongoing, the data obtained up to this date demonstrated that
ID8-injected mice had advanced stage tumors about 1 month after the injection (Figure
16 A) ,and that the vaccine, even without cytokine, conferred protection in terms
of survival advantage (Figure 16 B). These results show a survival advantage conferred
by an oral CTA-containing vaccine composition administered therapeutically even after
a short time. It is anticipated that as the experiment continues and the data mature
inclusion of cytokines will show a survival benefit or a reduction in tumor burden
or both. (In humans, prolongation of survival can be assessed by reference to mean
survival time from diagnosis).
[0118] The foregoing Examples are intended as illustrations and not limitations of the products
and methods disclosed herein.
[0119] All references cited herein are incorporated by reference in their entirety for all
purposes.
[0120] Aspects of the disclosure are set out in the following features:
- 1. A vaccine composition comprising:
- a. particles comprising nanoparticles, microparticles or a mixture thereof, the particles
comprising a gastric acid resistant, enteric erodable matrix comprising beta cyclodextrin
and hydroxypropylmethyl cellulose acetate succinate (HPMCAS), the particles encapsulating
one or more cancer testis antigens (CTA), and aleuria aurantia lectin (AAL), the AAL
being present in a microfold cell (M- cell) targeting effective amount; and
- b. an aqueous vehicle in which said particles are suspended, wherein the one or more
CTA is present in said vaccine composition in an amount effective to stimulate, upon
oral administration of said vaccine composition to a subject having a malignant tumor
expressing the one or more CTA, an immune response against said tumor.
- 2. The vaccine composition of feature 1 wherein the CTA is provided in an amount within
the range from about 1 to about 10% w/w based on the weight of the particles.
- 3. The vaccine composition of feature 1 wherein the one or more CTA is selected from
the group consisting of SP17, AKAP4, PTTG1, and Ropporinl and combinations of two
or more of the foregoing.
- 4. The vaccine composition of feature 1 wherein the one or more CTA is selected from
the group consisting of ASP, MAGE, NY-ESO-1, HER2neu, and Hml .24 and combinations
of two or more of the foregoing.
- 5. The vaccine composition of feature 1 the one or more CTA is selected from the group
consisting of SP17, AKAP4, PTTG1, and Ropporinl, ASP, MAGE, NY-ESO-1, HER2neu, and
Hml .24 and combinations of two or more of the foregoing.
- 6. The vaccine composition of feature 1 wherein the AAL is provided in an amount within
the range from about 0.1 to about 0.5% (w/w) based on the weight of the particles.
- 7. The vaccine composition of feature 1 further comprising at least one adjuvant.
- 8. The vaccine composition of feature 7 wherein the at least one adjuvant has the
property of stimulating at least a Th1 cellular immune response.
- 9. The vaccine composition of feature 8 wherein said at least one adjuvant has the
property of stimulating both a Th1 and a Th2 immune response.
- 10. The vaccine composition of feature 7 wherein said at least one adjuvant comprises
a first adjuvant for stimulating at least a Th1 immune response and a second adjuvant
for stimulating a Th2 immune response.
- 11. The vaccine composition of feature 7 wherein the at least one adjuvant is selected
from the group consisting of CpG, MF59, alum, flagellin, R848, monophosphoryl lipid
A, ODN 1826, and combinations of at least two of the foregoing.
- 12. The vaccine composition of feature 8 wherein the adjuvant comprises CpG.
- 13. The vaccine composition of feature 9 wherein the CpG is present in an amount within
the range from about 0.1 to about 0.5 % (w/w) based on the weight of the particles.
- 14. The vaccine composition of feature 7 wherein the adjuvant is provided in an amount
within the range from about 0.1 to about 0.5 % (w/w) based on the weight of the particles.
- 15. The vaccine composition of feature 1 or 7 further comprising at least one immune
response stimulating cytokine.
- 16. The vaccine composition of feature 7 wherein the at least one cytokine is selected
from the group consisting of interleukin 2 (IL-2), interleukin 12 (IL-12) or both.
- 17. The vaccine composition of feature 1 further comprising a lysate from said tumor.
- 18. The vaccine composition of feature 14 wherein the lysate is present in an amount
providing a lysate protein content (as measured by BCA assay) to the particles within
the range from about 1 to about 10 % (w/w) based on the weight of the particles.
- 19. The vaccine composition of feature 14 wherein the lysate has been prepared by
a process comprising use of hypotonic lysis buffer.
- 20. The vaccine composition of feature 1 wherein the CTA and the AAL are embedded
in the matrix.
- 21. The vaccine of composition feature 7 wherein the CTA, the AAL and the adjuvant
are embedded in the matrix.
- 22. The vaccine composition of feature 15 wherein the CTA, the ALL, the adjuvant and
the cytokine are embedded in the matrix.
- 23. The vaccine composition of feature 1 wherein the particles have an average diameter
based on particle volume within the range from about 1 to about 5 micrometers.
- 24. The vaccine composition of feature 1 wherein the nanoparticles have a normal size
distribution with at least 90 percent of the particles having an average diameter
between about 0.5 and 5 micrometers.
- 25. The vaccine composition of feature 1 wherein the nanoparticles have a zeta potential
within the range from about +50 to about -50 mV.
- 26. An assembly of particles comprising nanoparticles, microparticles or mixtures
thereof for use in a vaccine composition comprising:
- a. a polymer matrix comprising beta cyclodextrin and hydroxypropylmethyl cellulose
acetate succinate (HPMCAS),
- b. one or more cancer testis antigens (CTA);
- c. aleuria aurantia lectin (AAL),
and optionally one or more of the following;
d. an immune response stimulatory cytokine;
e an adjuvant; and,
f. tumor lysate antigens.
- 27. An assembly of particles according to feature 26 wherein the matrix comprises
60% beta cyclodextrin.
- 28. An assembly of particles according to feature 26 wherein the matrix comprises
30% HMPCAS and 15% EC.
- 29. An assembly of particles according to feature 26 wherein the CTA is one or more
antigens selected from the group consisting of SP17, AKAP4, PTTG1, Ropporin, ASP,
MAGE, NY-ESO-1, HEPv2neu, and Hml .24 and combinations of two or more of the foregoing.
- 30. An assembly of particles according to feature 26 wherein the adjuvant is selected
from the group consisting of CpG, MF59, alum, flagellin, R848, monophosphoryl lipid
A, ODN 1826, and combinations of at least two of the foregoing.
- 31. An assembly of particles according to feature 26 wherein the cytokine is at least
one cytokine that has the property of stimulating at least one of Th1 and Th2 immune
responses.
- 32. An assembly of particles according to feature 26 wherein the cytokine is one or
both of IL-2 and IL-12.
- 33. An assembly of particles according to feature 26 further comprising a lysate from
the tumor of a subject suffering from a malignant tumor.
- 34. An assembly of particles according to feature 26 having an average diameter based
on particle volume within the range from about 1 to about 5 microns.
- 35. An assembly of nanoparticles according to feature 26 having a normal size distribution
with at least 90 percent of the particles having an average diameter between about
0.5 and 5 micrometers.
- 36. An assembly of nanoparticles according to feature 26, wherein the nanoparticles
have a zeta potential within the range from about -50 to about +50 mV.
- 37. An assembly of nanoparticles according to feature 26 wherein the CTA is provided
in an amount effective, upon oral administration of the nanoparticles in a vaccine
composition to a subject having a malignant tumor expressing said at one or more CTA,
to stimulate an immune response against the tumor.
- 38. The assembly of nanoparticles according to feature 26 wherein the AAL is provided
in an amount effective to recruit microfold cells of said subject.
- 39. The assembly of nanoparticles according to feature 26 wherein the at least one
cytokine is IL- 2 and IL-12.
- 40. The assembly of nanoparticles according to feature 26 wherein the at least one
adjuvant is CpG.
- 41. A method for treating cancer comprising administering to a subject afflicted with
a malignant tumor a vaccine composition comprising
- a. particles comprising microparticles, nanoparticles or mixtures thereof, wherein
the particles comprise an enteric erodable matrix comprising beta cyclodextrin, hydroxypropylmethyl
cellulose acetate succinate (HPMCAS), the nanoparticles carrying one or more cancer
testis antigens (CTA) expressed in the tumor, and an M cell targeting lectin, aleuria
aurantia lectin (AAL), the AAL being present in a microfold cell targeting effective
amount; and
- b. an aqueous vehicle in which said particles are suspended, wherein the CTA is present
in said vaccine composition in an amount effective to stimulate, upon oral administration
of said vaccine composition to a subject having a malignant tumor, an immune response
against said tumor, and thereby treating the tumor.
- 42. A method according to feature 41 comprising repeating the administration at least
two and up to 10 times at an interval of 1 to 3 days after the preceding administration.
- 43. The method of feature 41 wherein the treatment results in one or more of reducing
tumor burden in said subject and prolonging survival of said subject
- 44. A method according to feature 41 wherein the subject is afflicted with a malignant
tumor selected from the group of solid tumors and hematologic malignancies.
- 45. The method of feature 41 wherein the tumor is a solid tumor selected from the
group consisting of ovarian cancer, hepatocellular carcinoma, breast cancer, and prostate
cancer.
- 46. The method of feature 41 wherein the tumor is a hematologic malignancy selected
from the group consisting of multiple myeloma, acute myelogenous leukemia, acute lymphoblastic
leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia.
- 47. The method of feature 41 comprising administering to the subject a vaccine composition
in an amount within the range between about 5mg to 320 mg said composition containing
from about 1 to about 10 % (w/w) of CTA, fromabout 0.1 to about 0.5 % (w/w) of AAL
based on the weight of the particles.
- 48. The method of feature 41 wherein the tumor is ovarian cancer.
- 49. The method of feature 41 wherein the CTA is SP17.
- 50. The method of feature 41 wherein the composition further comprises an adjuvant.
- 51. The method of feature 41 wherein the composition further comprises at least one
immunostimulatory cytokine is two cytokines: IL-2 and IL-12.
- 52. The vaccine composition of feature 41 wherein the CTA is one or more of MAGE-A3,
MAGE-A9 and MAGE-A12.
- 53. The assembly of feature 26 wherein the CTA is one or more of MAGE -A3, MAGE-A9
and MAGE-A12.
- 54. The vaccine composition of feature 1 wherein the matrix also comprises ethyl cellulose.
- 55. The assembly of feature 26 wherein the matrix also comprises crosslinked ethyl
cellulose.
- 56. The vaccine composition of feature 1 wherein the matrix comprises 60% beta cyclodextrin,
20% HPMCAS and 15% ethyl cellulose.
- 57. The assembly of feature 26 wherein the matrix comprises 60% beta cyclodextrin,
20%> HPMCAS and 15% ethyl cellulose.
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